Friedreich ataxia is the most common inherited ataxia. Friedreich ataxia patients have large expansions of a GAA triplet-repeat sequence in both copies of the FXN gene. The progressive ataxia is mainly "sensory" and is due to primary degeneration of dorsal root ganglia (DRG). The cause for the selective and progressive degeneration of the sensory DRG neurons is not known. A sensitive method called small pool PCR to analyze somatic instability of the expanded GAA triplet-repeat sequence revealed that patients have further large expansions in their DRG, which accumulated in an age-dependent fashion. Moreover, in a "humanized" transgenic mouse model carrying an expanded GAA triplet-repeat sequence within the context of the entire human FXN gene, the same age-dependent increase in prevalence of large expansions in the DRG was observed. The goal of this project is to evaluate the role of this somatic instability in Friedreich ataxia. We hypothesize that these large expansions occur specifically in neurons, and based on preliminary data, that the underlying mechanism involves DNA double-strand breaks (DSBs). We will use single-cell analysis of neurons and glial cells from DRG of two humanized transgenic mouse models to investigate what cell-type is responsible for the large expansions observed in whole-tissue preparations. DRG neurons obtained from autopsy tissue of multiple Friedreich ataxia patients will be similarly analyzed. To test the hypothesis that DSBs are associated with tissue-specific somatic instability, co-localization studies with immunofluorescence and FISH, and chromatin immunoprecipitation experiments will be used to identify DSBs at the site of the expanded GAA triplet- repeat sequence in vivo. The effect of DSBs on GAA repeat instability will be directly tested in cell culture experiments by either using repair-deficient cell lines, or treating cells from patients with DSB-inducing drugs. Finally, to test the hypothesis that the expanded GAA triplet-repeat is inordinately susceptible to DSBs, we will cross the existing mouse models for somatic instability on to a repair-deficient, ATM null background. Our data will have important implications for understanding the pathogenesis of Friedreich ataxia. Additionally, if a mechanism for the somatic instability is identified, this may yield new clues for the development of a specific therapy to slow disease progression. PUBLIC HEALTH RELEVANCE: Patients with Friedreich ataxia, the most common inherited ataxia, have expanded GAA repeat sequences in their FXN genes. Preliminary results indicate that instability of this sequence is important for the pathogenesis and progression of disease. Our experiments are designed to confirm this notion, and to determine the mechanism of this DNA instability.